Tissue spectroscopic characterization based on fluorescence, second harmonic generation, and reflected light

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2004."September 2004."Includes bibliographical references (leaves 81-92).The diagnosis of many diseases often requires a histological analysis of tissues. Histology analysis compares the microscopic structure of a tissue specimen with an image database containing known physiological and pathological tissue structures. Three new microscopy technologies are developed to complement histology based on novel contrast mechanisms to better visualize and understand tissue structure and function: two-photon spectral resolved imaging, tri-modal imaging, and interferometric second harmonic imaging. First, two-photon spectral resolved microscopy utilizes the 3D localization ability of two-photon excitation to extract spectroscopic information from a femtoliter volume in tissue. The method is capable of the identification of biochemical species in tissues based on their morphological and spectral signatures. This system incorporates two new spectral analysis methods - spectral image guided analysis and multivariate curve resolution. This instrument has been applied to the study of human skin luminescence species and in a photoaging study of a skin equivalent model. Second, tri-modal microscopy combines two-photon fluorescence with second harmonic imaging and reflected light optical coherence microscopy. In this tri-modal system, fluorescence imaging maps fluorophore distribution; second harmonic imaging maps biological crystalline structures such as collagen and microtubules; reflected light optical coherence microscopy maps index of refraction heterogeneity. The ability of this tri- modal microscope has been demonstrated in the imaging of black tetra fish scale and in ex vivo human skin. Third, interferometric second harmonic microscopy has the potential for imaging deeper second harmonic active structures in tissues.(cont.) This enhancement is based on phase coherent detection allowing the separation of multiple scattered light from the ballistic second harmonic signal. We have implemented interferometric second harmonic microscopy in epi-imaging mode and demonstrated coherent imaging of non-linear optical crystals.by Lily H. Laiho.Ph.D

Investigating the Protective Properties of Milk Phospholipids Against Ultraviolet Light Exposure in a Skin Equivalent Model

Current research on bioactive molecules in milk has documented health advantages of bovine milk and its components. Milk Phospholipids, selected for this study, represent molecules with great potential benefit in human health and nutrition. In this study we used confocal reflectance and multiphoton microscopy to monitor changes in skin morphology upon skin exposure to ultraviolet light and evaluate the potential of milk phospholipids in preventing photodamage to skin equivalent models. The results suggest that milk phospholipids act upon skin cells in a protective manner against the effect of ultraviolet (UV) radiation. Similar results were obtained from MTT tissue viability assay and histology

Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra

Spectral resolved tissue imaging has a broad range of biomedical applications such as the minimally invasive diagnosis of diseases and the study of wound healing and tissue engineering processes. Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. While two-photon excited autofluorescence is observed ubiquitously, the identities and distributions of endogenous fluorophores have not been completely characterized in most tissues. We develop an image-guided spectral analysis method to analyze the distribution of fluorophores in human skin from 3-D resolved two-photon images. We identify five factors that contribute to most of the luminescence signals from human skin. Luminescence species identified include tryptophan, NAD(P)H, melanin, and elastin, which are autofluorescent, and collagen that contributes to a second harmonic signal.Unilever (Firm)American Cancer Society (RPG-98-058-01-CCE)National Institutes of Health (U.S.) (R33 CA091354-01A1